Hindawi Publishing Corporation ISRN Spectroscopy Volume 2013, Article ID 230858, 12 pages http://dx.doi.org/10.1155/2013/230858

Review Article A Recent Review on Chemiluminescence Reaction, Principle and Application on Pharmaceutical Analysis

Tadesse Haile Fereja,1 Ariaya Hymete,2 and Thirumurugan Gunasekaran1

1 DepartmentofPharmacy,CollegeofMedicineandHealthSciences,AmboUniversity,Ambo19,Ethiopia 2 Department of Pharmaceutical Chemistry and Pharmacognosy, Addis Ababa University, Addis Ababa 117, Ethiopia

Correspondence should be addressed to Tadesse Haile Fereja; tdss [email protected]

Received 16 July 2013; Accepted 15 September 2013

Academic Editors: V. M. Bermudez, H. J. Byrne, and Z. Wang

Copyright © 2013 Tadesse Haile Fereja et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

This paper provides a general review on principle of chemiluminescent reactions and their recent applications in drug analysis. The structural requirements for chemiluminescent reactions and the different factors that affect the efficiency of analysis are included in the review. Chemiluminescence application in immunoassay is the new version for this review. Practical considerations are not included in the review since the main interest is to state, through the aforementioned applications, that chemiluminescence has been, is, and will be a versatile tool for pharmaceutical analysis in future years.

−13 1. Introduction of the exited state is lost, and this process occurs in 10 −11 to 10 sec. This means that before an exited molecule in Luminescence is the most conveniently defined as the radi- a solution can emit a photon, it will undergo vibrational ation emitted by a molecule, or an atom, when these species relaxation, and therefore photon emission will always occur return to the ground state from the exited state. According to from the lowest vibrational level of an excited state [3, 4]. the source of excitation, luminescence phenomenon could be Once a molecule arrives at the lowest vibrational level of classified as photoluminescence (fluorescence and phospho- an exited singlet state, it can do a number of things, one of rescence) when the excitation source is energy from absorbed whichistoreturntothegroundstatebyphotonemission. light, chemiluminescence-energy from chemical reactions This process is called fluorescence. The lifetime pf an exited −9 −7 and bioluminescence energy from biologically catalysed reac- singlet state is approximately 10 to 10 sec and therefore the tions. An exited molecule has the same geometry and is in the decay time of fluorescence is of the same order of magnitude. same environment as it was in ground state. In this situation it The quantum efficiency of fluorescence is defined asthe either emits a photon from the same vibrational level to which fraction of molecules that will fluoresce. it was exited initially or it undergoes in vibrational level prior In addition to fluorescence, one also encounters radia- to emission of radiation [1, 2].Foranisolatedmoleculeinthe tionless process where molecules in an excited singlet state gas phase, the only way to lose vibrational energy is to emit mayreturntothegroundstatewithouttheemissionofa an infrared photon, which is less probable than undergoing an photon, converting all the excitation energy into heat: the electronic transition to return to the ground state. Therefore, process called internal conversion. Generally, internal con- onetendstoseephotonemissionfromhighervibrational version is an inefficient process and is probably only a small levels of exited states in gas phase spectra at low pressure. In a fraction of the total excitation energy in most molecules. solution, however, thermal relaxation of a vibrationally exited Although population of triplet states by direct absorption molecule is quite rapid through transfer of excess vibrational from the ground state is insignificant, a more efficient process energyfromthesolutemoleculetothesolvent.Infact,this exists for population of triplet states from the lowest excited process is so efficient that all the excess vibrational energy singlet state in many molecules. This process is referred 2 ISRN Spectroscopy

Singlet excited states Triplet excited state Internal Vibrational conversion relaxation S2 Intersystem crossing

S1 T1

Internal Energy and Fluorescence Absorption external Phosphorescence conversion

S Vibrational 0 relaxation Ground state 𝜆 󳰀 𝜆 𝜆 2 𝜆1 𝜆r r 3 𝜆4

Figure 1: Jablonski diagram describing the electronic levels of common organic molecules and possible transitions between different singlet and triplet states. (M. Sauer, J. Hofkens, and J. Enderlein. Handbook of Fluorescence Spectroscopy and Imaging).

to as intersystem crossing and is a spin-dependent internal electronically excited molecule, responsible for light emission conversion process. A molecule in excited triplet state may or acting as the energy as transfer donor in indirect CL. The not always use intersystem crossing to return to the ground catalyst enzyme or metal ions reduce the activation energy state. It could loss energy by emission of a photon: the process and provide an adequate environment for producing high called phosphorescence. A triplet-singlet transition is much CL efficiency out of the process. Cofactors sometimes are less probable than a singlet-singlet transition. The lifetime of necessary to convert one or more of the substrates into a form the excited triplet state can be up to 10 sec, in comparison capable of reacting and interacting with the catalyst, or to −5 −9 with 10 sto10 s of average lifetime of an excited singlet provide an efficient leaving group if bond cleavage is required state. Emission from triplet-singlet transition can continue to produce the excited emitter. On the contrary, indirect or after initial irradiation19 [ , 20]. sensitized CL is based on a process of transfer of energy of the excited specie’s to a fluorophore23 [ ]. This process makes 2. The Nature of Chemiluminescence it possible for those molecules that are unable to be directly involved in CL reaction to transfer their excess of energy to Reactions a fluorophore that in turn is excited, releasing to its ground state with photon emission. All of these paths lead to a great In general, chemiluminescence reaction yields one of the variety of practical uses of CL in solid, gas, and liquid phases reaction products in an electronic excited state producing [24]. light on falling to the ground state. As can be seen in Figure 1, theprocessoflightemissioninchemiluminescenceisthe same as in photoluminescence, except for the excitation pro- 2.1. Requirements for Chemiluminescence Emission. For a cess. In fluorescence and phosphorescence, the electronically chemical reaction to produce light, it should meet some excited state is produced by absorption of Uv-visible light essential requirements. returning to the ground state (S0) from the lowest singlet (1)Thereactionmustbeexothermictoproducesufficient excited state (S1) or from the triplet excited state (T1)[21, 22]. energy to form the electronically excited state. To In general, a chemiluminescent reaction can be generated predict whether the CL reaction will occur or not, it by two basic mechanisms (Figure 2) in a direct reaction, ispossibletousefreeenergy(Δ𝐺): two reagents, usually a substrate and an oxidant in the presence of some cofactors, react to from a product or Δ𝐺 = Δ𝐻 − 𝑇Δ𝑆, intermediate, sometimes in the presence of a catalyst. Then (1) some fraction of the product or intermediate will be formed in an electronically excited state, which can subsequently relaxtothegroundstatewithemissionofaphoton.The where 𝑇 is temperature, Δ𝐻 is enthalpy, and Δ𝑆 is substrate is the CL, precursors, which is converted into the entropy. ISRN Spectroscopy 3

A +B+(Cofactors) (Chemiluminescent (Oxidant) AB + light precursor) D A + B 2 AB + D ( ) Catalyst (7)

∗ Direct P (intermediate or product) chemiluminescence ∗ Senitized or energy transfer BA AB AC +B chemiluminescence F 3 (6) ( ) C P +h P +h 5 4 F ( ) ( ) +h ∗ AB+ F AB + F

Figure 2: Types of CL reaction, P, product F, fluorescing substance. Figure 3: different processes for losing energy from the excited state: (1) direct CL, (2) molecules dissociation; (3) chemical reaction with other species, (4) intramolecular energy transfer, (5) intermolecular Enthalpy is the actual energy source for generating energy transfer (in case of a fluorphore, indirect CL), (6) isomeriza- the electronically excited state product. To initiate tion, and (7) physical quenching. the chemical reaction, the activation energy (Δ𝐻𝐴) is absorbed. The available energy to produce the Δ𝐸 (3) Photon emission must be a favorable deactivation excited state ( EX) must be the difference between the reaction energy and the activation energy. If the process of the excited product in relation to other difference is equal to or greater than the energy competitive nonradiative processes that may appear Δ𝐸 in low proportion (Figure 3) in the case of sensitized required for generating the excited state EX,theCL process will be produced. Consider CL, both the efficiency of energy transfer from the excited species to the fluorophore and the fluroesence efficiency of the latter must be important. =Δ𝐻 −Δ𝐻 ≥Δ𝐸 . In all the luminescent process, the intensity of the Energy Available 𝐴 𝐸 EX (2) produced emission depends on the efficiency of generating molecules in the excited state, which is represented by the quantum efficiency (quantum yield) and the rate of the In many CL reactions, the entropy change is small, so reaction. In the case of CL reactions, the intensity can be Δ𝐺 and Δ𝐻 are very similar in magnitude and the expressed as energetic requirements can be established in terms −1 𝐴 of (Kcal⋅mol ). In this sense, for CL to occur, the 𝐼 = ́ − d , CL ØCL (4) reaction must be sufficiently exothermic such that d𝑡 𝐼 ́ where CL is the CL emission intensity (photons/second) ØCL is the CL quantum yield and (−d𝐴/d𝑡) is the rate at which ℎ𝑐 2.86 × 104 𝐴 Δ𝐺 ≥ = , the CL precursor is consumed. Higher values of quantum 𝜆 𝜆 (3) yields are usually associated with BL reactions whereas in ex ex mostoftheCLreactionsusedforanalyticalpurposesrange from0.001to0.1andevenveryinefficientsystemswithmuch 𝜆 lowerquantumyieldscanbeusedinanalysisbasedonthe where ex is the long wavelength limit (nanometers) forexcitationoftheluminescentspecies.Because complete absence of background emission. This is the case with ultraweak CL reactions, in which can be less than 0.001 most of the CL reaction produce photons in the −3 −2 −9 −15 range 400 (violet)–750 (red) nm, the creation of the (often 10 –10 ) and sometimes 10 –10 . Ultra-weak CL is produced from oxidative reaction in electronically excited state and the generation of CL in 3 6 −1 living cells and the emitted signals are mostly 10 –10 times the visible region require around 40–70 Kcal⋅mol . less intense than those from luminous organisms. The main This exothermic condition is associated with redox characteristic of ultraweak CL emission is that it is invisible reactionsusingoxygenandhydrogenperoxideor to the naked eye. This kind of CL includes a group of CL similar potential oxidants [25]. reactions that at least in living cells, involve a number of (2) The reaction pathway must be favorable to channel oxygen intermediate that play an important role in certain the energy for the formation of an electronically types of cell activation in the defense system of the body, and excited state. In case the chemical energy is lost as in ischemic heart diseases. It has been detected from a wide heat, for example via vibration and rotational energy variety of infact organs, isolated cells, and tissue homogenates ways, the reaction will not be chemiluminescent. from vertebrates, invertebrates, plants, and from several 4 ISRN Spectroscopy reactions in vitro.Ultra-weakCLisassociatedwithsome important cellular functions, such as mitochondrial respira- tions, photosynthesis, cell division, or phagocytosis, among others [26–28]. ΔH 2.2. Some Factors Influencing Chemiluminescence’s Emission. Because of the cited dependence of CL intensity upon ΔH

various parameter, CL measurement’s are strongly modified Potential energylevel by experimental factors that affect quantum yield and rate of reaction, such as Distance between nuclei(r) (i) the chemical structure of the CL precursor, not only the central portion containing the electronically Figure 4: Relative positions of the potential energy (𝐸) surface of excited group, but also the side chain; the electronic states involved in a hypothetical chemiluminescent reaction as a function of internuclear separation (𝑟). (ii) the nature and concentration of other substrates affecting the CL pathway and favoring other nonra- diative competition process; (iii) the hydrophobicity of the solvent and solution com- at lower energy than when the dark product in the ground position as example the of luminal oxidized in electronic state hase the same configuration as the reactants dimethylusfoxide (DMSO) is 0.05 compared with 0.01 in their ground electronics state. This is illustrated in Figure 4. in water, the colors being blue-violet (425 nm) and Often,catalysts,whichareusuallytransitionmetalionsin blue green (480–502 nm), respectively; chemilluminercent reaction and enzymes in bioluminescent (iv)thepresenceofenergytransferacceptors[29]. reactions, are employed to make light emitting reactions processed at a convenient rate. This is accomplished by the catalyst forming a transient complex with reagents. Whose 2.2.1. Chemical Considerations. AsinequanonforCLto Δ𝐻4𝑖, is lower than that of unanalyzed reaction. The catalyst occur is that the precursor(s) of the light-emitting species is usually a cooxidant; that is, it assists electron transfer. must participate in reaction that releases a considerable The catalysis of the luminol hydrogen peroxide reaction by amount of energy. For visible emission (say 400–750 nm Co(II) and of the luciferian-oxygen reaction by the enzyme in wavelength), 40–70 kcal⋅mol is required. Actually, the luciferase are important examples (6,13). ∗ enthalpies of reaction have to be slightly greater than these 𝑝 and 𝑝 represent the ground and lowest electronically values owing to energy losses by thermal relaxation in the excited singlet states of the product of the reaction, respec- ground state (after emission) and lowest excited singlet (after tively, and 𝑅 represents the ground electronic state of the excitation) states of the fluorophore. Normally only certain reactant. Δ𝐻 is the enthalpy of the “dark” reaction while oxidation-reduction reactions generate this much energy. Δ𝐻4 is the enthalpy of activation. Δ𝐻4 is the enthalpy of In addition, at least some of the energy produced must activation of the photoreaction hv that denotes the emission be channeled into a reactions pathway, in which at least of chemiluminescence [32]. one of the upper vibration levels of the reactants, probably corresponding to the transition state of the initial reaction, has the same energy and a comparable geometrical structure 2.2.2. Photophysical Considerations. Subsequent to the for- asanuppervibrationallevelofthelowestexcitedsinglet mation of the potentially CL molecule in its lowest excited state of potentially emissive product of the reaction. The singlet state, a series of events carry the excited molecule geometric identity or near-identity of the interconverting down to its ground electronic state. Since the electronically species ensures that most of the energy of activation is excited molecule is initially also vibrationally excited, rapid, −13 −12 channeledintofreeenergyofactivationratherthanbeing stepwise (10 –10 s) thermal deactivation of the excited wasted as entropy or activation [30, 31]. molecule, called thermal or vibrational relaxation, in which Invariably, there will also be pathway for dark reaction. the molecule loses vibrational energy by inelastic collisions The dark reaction may lead directly to the ground, electronic with the solvent occurs. The process of vibrational relaxation state species that also results from the fluorescence of the carries the molecule to the lowest vibrational level of the excited product, but this is not necessarily so. It is possible to lowest excited singlet state. Certain molecules may return have a competing dark reaction that leads directly to ground radiationlesly all the way to the ground electronic state in the state products different from that produced by fluorescence. same time frame by thermal deactivation, a process known as If Δ𝐻4, is the sum of the enthalpies of activation for all dark internal conversion. However, in some molecules, for a vari- reaction competing with the chemilluminercent pathway, ety of reactions, the return from the lowest vibrational level of whose enthalpy of activation is Δ𝐻4𝑖 CL will be probable the lowest excited singlet state to the lowest vibrational level when Δ𝐻4 <Δ𝐻4𝑖 (3,8). This occurs when the lowest excited of the ground state by internal conversation and vibrational singlet state of the flurophore has the same geometrical relaxation is forbidden (i.e., of low probability or long configuration as the ground electronic state of the reactants duration). In these molecules, return to the ground electronic ISRN Spectroscopy 5 state occurs by one of two alternative pathways, the simpler The less vibrational and rotational freedom that the molecules being the direct emission of ultraviolet or visible radiation has, the greater is the probability that the energy gap between whose frequency or wavelength is governed by the energy the lowest excited singlet state and the ground electronic gap between the lowest excited single state and the ground statewillbelarge,sothatfluorescencewillpredominate electronic state [33]. The radiative transition between excited over nonradiative deactivation and assure a high fluorescence andgroundstateofthesamespinmultiplicityoccursinatime efficiency. −11 −7 frame 10 –10 safterexcitation,andiscalledfluorescence. Aromatic molecules containing freely rotating sub- Owing to the fact that the ground electronic state of molecule stituents usually tend to fluoresce less intensely than those has several vibrational levels associated with it, fluorescence without these substituents. These result from the introduction emission does not occur at a single wavelength but rather over into each electronics state of rotational and vibrational a range of wavelength corresponding to several vibrational substatesbytheexocyclicsubstituents[37]. transitions as components of a single electronic transition [34]. 2.2.5. The Influence of the Environment on Several processes may compete with fluorescence for the Fluorescence Spectrum deactivation of the lowest excited singlet state. As a result, only a fraction of the molecules formed in the lowest excited singlet state. Ø Actually fluoresce. Ø is called the quantum Solvent Effect. Solvent interactions with molecules are mainly yield or fluorescence efficiency. It is usually a fraction but electronic and it is usually the difference between the may be unity in some exceptional cases and is related to electrostatic stabilization energies of ground and excited 𝑘 the probabilities (rate constant) of fluorescence ( ef)and state that contribute to the relative intensities and spectral competitive process (𝑘𝑑)[35]. positions of fluorescence in different solvents. The change in electron distribution that takes place upon transition from 2.2.3. Structural and Environmental Consideration. the lowest excited singlet state to the ground state during Molecules that enter into CL reaction are generally reduced fluorescence causes changes in the dipolar and hydrogen- species that can be easily oxidized. Molecules containing bonding properties of the solute. If the solute is more polar amino and hydroxyl groups fall into this category, as do in the excited state than in the ground state, fluorescence polycyclic aromatic ring systems. Under strongly alkaline willoccuratlongerwavelengthinapolarsolventthanin conditions, hydroxyl groups and even some arylamino or a nonpolar solvent. This is because, the more polar solvent arylamido groups can be deprotonated making them even will stabilize the excited state relative to the ground state. more susceptible to oxidation (Table 1). On the other hand, Moreover, the fact that photoluminescence originates from electron-withdrawing groups tend to stabilize electronic an excited state that is in equilibrium with its solvent cage and charge and make oxidation more difficult in molecules terminates in a ground state that is not cause the fluorescence to which they are affixed. Form the chemical point of to lie at longer wavelengths the more polar or more strongly view, the solvent in which the CL experiment is carried hydrogen-bonding the solvent [38]. out can have a dramatic influence on the efficiency of the The lowest excited singlet state is formed by promoting a ∗ CL reaction as salvation can alter the shapes, the depths, lone-pair electron to a vacant 𝜋 orbital (this is called an 𝑛, 𝜋 and the densities of the vibrational states of the potential state). These molecules tend to show very little fluorescence surfaces representing the ground states of products and inaproticsolventssuchasaliphatichydrocarbonsbecause ∗ reactants and the lowest excited singlet state of the potential the 𝑛, 𝜋 excited singlet state is efficiently deactivated by fluorophore. The alteration of the intersections of these intersystem crossing. However, in portic solvents such as potential energy surface an affect the enthalpies of reaction water to ethanol, these molecules become fluorescent. This ∗ and the enthalpies of activation for dark and lumigenic results from the destabilization of the lowest singlet 𝑛, 𝜋 state ∗ reactions. In some cases, these changes will favor CL (if Δ𝐻 by hydrogen bonding. If this interaction is sufficiently strong, ∗ ∗ ∗ decreases relative to Δ𝐻 ) and in some cases, they will make the fluorescent 𝜋, 𝜋 state drops below the 𝑛, 𝜋 state allowing it thermodynamically unfavorable for CL to occur. Other intense fluorescence. Quinoline, for example, fluoresces in influences of the structure and the environment are manifest water but not in cyclohexane [39]. in the rates of processes competing with fluorescence for Solvents containing atoms of high atomic number (e.g., deactivation of the lowest excited singlet state. These are alky iodide or bromides) also have a substantial effect on the the process and properties that influence the fluorescence intensity of fluorescence of solute molecules. Atoms of high process [36, 37]. atomicnumberinthesolventcageofthesolutemolecule enhance spin-orbital coupling in the lowest excited singlet 2.2.4. The Influence of Molecular Structure on Fluorescence. state of the solute. This favors the radiationless population Among other factors, the quantum yield of fluorescence of the lowest triplet state at the expense of the lowest excited determines the intensity of lights emission in a CL. This, as singlet state. Thus in heavy-atom solvents, all the other things well as the position in the spectrum occupied by the fluores- being equal, fluorescence is always less intense than that in cence band, is largely a function of the molecular structure. solvent of low molecular weight [40]. Fluorescence is most often observed in highly conjugated or Quenching of Fluorescence. Fluorescence may be decreased aromatic organic molecules with rigid molecular skeletons. or completely eliminated by interactions with other chemical 6 ISRN Spectroscopy

Table 1: Common application of the main CL System.

Phase Reagents Analyte References

Gas Ethylene O3 [5] O after conversion of the analyte Gas 3 Nitrosamine total nitrogen [6] to NO

Gas H2 flame Sulfer compounds7 [ ]

Gas H2 flame followed by O3 Sulfer compounds [7] Metal ions and complexes (Co(II), Cu(II), Fe(III), Zn(I), Cd(II), − −3 Mn(II), Cr(III), Cr(Iv), Pt(Iv), ClO Fe(CN)6 , Heme compounds, Peroxides, oxidants (H2O2,O2,I2 etc...), Inhibitors (Ag(I), Ce(IV), Ti(IV) [8–13] Liquid Luminal and derivatives Substance easily oxidized and indirectly determined (Ascorbic acid, carboxylic acids, amines, etc...) ... Substances converted in to H2O2 (glucose, etc ) Substances labeled with luminal and derivatives Substance labelled with acridinium esters, Ions Ag(I), Bi(III), Pb(II), Co(II), Cr(III), Cu(II), Fe(III), Mn((II), etc...) ... Liquid Acridinium esters Oxidants (H2O2,O3 etc ), Substances converted in to H2O2 [14, 15] Reducing compounds (Cr(II), Fe(II), Mo(V), ascorbic acids, tetracyclines sugars, etc...) ... Oxidants (H2O2,O3 etc ), Flourophores (polycyclic aromatic hydrocarbons, etc...), Derivativatized compounds with fluorophores Liquid Peroxyoxalates [10, 12] (amino acids, steroids, aliphatic amines, carboxylic acids, catecholamines, etc...) Direct oxidation with MnO , Liquid − − 4 Different molecules (usually in pharmaceutical applications)16 [ –18] ClO , Ce(Iv), IO4 etc

species. This phenomenon is called quenching of fluores- to be observed (say1 % quenching), [𝑄]mustbegreaterthan −4 cence. Obviously, if the fluorescence of fluorophore generated or equal to 10 M[41, 42]. in CL reaction is quenched the observation of chemilumines- cencewillbeprecluded. Energy Transfer. Energy transfer entails the excitation of a Two kinds of quenching are distinguished. In static molecule that during the lifetime of the excited state passes quenching, interaction between the potentially fluorescent its excitation energy to other molecules. The loss of excitation molecule and the quencher takes place in the ground state, energy from the initial excited species (the door) results in forming a nonfluorescent complex. The efficiency of quench- quenching of the luminescence of the energy donor and may ing is governed by the formation constant of the complex as result in luminescence from the energy recipient (acceptor), well as by the concentration of the quencher. The quenching which becomes excited in the process. of the fluorescence of salicylic acid by Cu(II) is an example. Energy transfer can occur by either or two acceptor con- In dynamic quenching (or diffusional quenching) the centration dependent processes. In the resonance excitation quenching species and the potentially fluorescent molecule transfer mechanism or dipole mechanism, the donor and react during the lifetime of the excited state of the latter. The acceptor molecules are not in contact with one another and efficiency of dynamic quenching depends upon the viscosity may be separated by as much as 10 nm (although transfer of the solution, the lifetime of the excited state (£𝑞)ofthe distance closer to 1 nm are more common). In the classical of the luminescent species, and the concentration of the sense, the excited energy donor molecule may be thought quencher [𝑄]. This is summarized in the Stern-Volumer of as transmitting antenna that creates an electrical field in equation: its vicinity. Potential acceptor molecules within the range of this electrical field function as receiving antennae and absorb Ø 1 = , energy from the field resulting in their electronic excitation. 1+𝑘 ( ) 𝑄 (5) Ø𝑞 𝑄 £𝑞 [ ] The rate of resonance energy transfer decreases with the sixth powerofthedistancebetweenthedonorandacceptordipoles where 𝑘𝑄 is the rate constant for encounter between quencher according to and potentially luminescing spices and Ø and Ø𝑞 are the quantum yields of fluorescence in the absence and presence 1 𝑅 𝐾 = 0 , of concentration [𝑄] of the quencher, respectively. ET (6) £𝐷 𝑅 𝑘𝑄 Is typical of diffusion-controlled reactions 10 −1 −1 (∼10 M s ) white for a fluorescent molecule is typically where 𝐾 is the rate constant for resonance energy transfer, −8 2 −1 ET 10 or less. Hence, 𝑘𝑞 ≤ 10 M and for dynamic quenching and £𝐷 is the lifetime of the excited state of the donor ISRN Spectroscopy 7

́ molecule. 𝑅 is the mean distance between the center for where Ø𝑅 the chemical yield, is the ratio of the number the donor and acceptor dipoles, and 𝑅0 is constant for of molecules that react, through the chemilluminercent ́ a given donor-acceptor pair, corresponding to the main pathway to the total number of molecules reacted; Ø𝐸,the distance between the centers of the donor and acceptor excitation yield, is the ratio the number of molecules that dipoles for which energy transfer from donor to acceptor form an electronically excited product to the number of and florescence from the donor are equally probable. Another molecules that react through the Chemiluminescent pathway, ́ general requirement for the occurrence of resonance energy and Ø𝑓 is the quantum yield of fluorescence of the light- transfer is the overlap of the fluorescence spectrum of the emitting species. donor and the absorption spectrum of the acceptor. Any In type II reaction degree of overlap of these spectra will satisfy the quantization requirements for the energy or the thermally equilibrated ́ ́ ́ ́ ́ Ø = Ø𝑅 × Ø𝐸 × Ø𝑓 × Ø , (8) donor molecules to promote the acceptor to a vibrational level CL ET of excited singlet state. The greater the degree or overlap of where all symbols have the same meanings as above and the luminescence spectrum of the donor and the absorption ́ Ø is the efficiency of energy transfer from the initially spectrum of the acceptor, the greater is the probability that ET ́ energy transfer will take place [43, 44]. chemiexcited species to the energy transfer acceptor ØCL, The exchange mechanism of excitation energy transfer ofcourse, a function of the chemical and photo physical is important only when the electron clouds of donor and factors described in Section 2.2.2 such as solvent polarity, reagent concentrations, and molecular structure. acceptor are in direct contact. In this circumstance, the ́ highest energy electrons of the donor and acceptor may The physical significant of ØCL is the under defined exchange places. Thus, the optical electron of an excited experimental conditions it is the constant of proportionality donor molecule may become part of the electronic structure between, the observe intensity of chemiluminescence, and of an acceptor molecule originally in the ground singlet state the rate of consumption of the initial luminophore (reactant 𝑙 = ́ ×(− 𝐿/ 𝑡) in while the donor is returned to its ground singlet state by L); that is, CL ØCL d d [47, 48]. acquiring an electron from the acceptor. Exchange energy transfer is also the most efficient when the fluorescence 3. Chemiluminescence Based Analysis spectrum of the donor overlaps the absorption spectrum of the acceptor. Exchange energy transfer is a diffusion- Because the reaction rate is function of the chemical concen- controlled process (i.e., every collision between donor and tration, CL techniques are suitable for quantitative analysis. acceptor leads to energy transfer) and such its rate depends on The usefulness of CL system in analytical chemistry is based the viscosity, of the medium. Resonance energy transfer, on on some special characteristic. the other hand, is not diffusion-controlled, does not depend upon solvent viscosity and may be observed at the lowest (1) As the technique simultaneously comprises kinetic concentrations of acceptor species. Energy transfer from the and luminescence features, it provides highly sensi- initially chemiexcited species to a suitable acceptor flowed by tive and wide dynamic ranges. Excellent detection fluorescence from the acceptors in an important process in limits, in the range of femtomoles, can be reached if ́ chemiluminescence [45, 46]. ØCL is high enough. As an example, in the gas phase, typical detection limits are 10 pmol NO using ozone and 0.1 pmol for sulfur compounds using a hydrogen 2.3. Chemiluminescence Reaction Parameters. CL reaction is flame followed by ozone, in the liquid phase down commonlydividedintotwoclasses.InthetypeI(direct) to 1 fmol of the fluorophore may be detected using reaction the oxidant and reductant interact with rate constant 𝑘 the peroxyoxlalte CL system and 0.1 fmol peroxidase 𝑟 to directly from the excited product whose excited singlet when using luminol. In comparison to other spec- state decays with the first (or pseuodfrist)-order rate constant trometric techniques, in general, CL is approximately 𝑘 =𝑘 +𝑘 5 𝑟 𝑓 𝑑.IntypeII(indirect)reactiontheoxidantand 10 time more sensitive than absorption spectrometry reactant interact with the formation of an initially excited 3 and at least 10 times more sensitive than fluorimetry. product (𝑘)followedbytheformationofanexcitedsecondary product, either by subsequent chemical reaction or by energy (2) Compared to photoluminescence process, no external transfer, with constant 𝐾. The secondary product then decays light source is required, which offers some advantages from the lowest excited singlet state with rate constant 𝑘.Type such as the absence of scattering or background II reactions are generally denoted as a complex or sensitized photoluminescence signal, the absence of problems chemiluminescence. related to instability of the external source, reduction ́ If ØCL is the efficiency of the chemiluminescence reac- of interferences due to a nonselective excitation pro- tion, which is the ratio of the number of photons emitted to cess, and simple instrumentation. the number of molecules of reactant reacting it can be defined (3) Selectivity and linearity are most dependants on the foratypeIreactionas reaction conditions chosen. As for photolumines- cence process, absorption or emission radiation by ́ = ́ × ́ × ́ , the analyte, products, or concomitants can cause ØCL Ø𝑅 Ø𝐸 Ø𝑓 (7) nonlinearity or spectral interference. 8 ISRN Spectroscopy

(4) The technique is versatile for determination ofa using a micro liter plate reader or by contact printing with wide variety of species that can participate in the photographic detection. The chemilluminercent intensity of CL process, such as CL substrates or CL precursors thesesystemsisinfluencedbyboththekineticsofthereaction responsible for the excited state; the necessary reagent and the efficiency of the mass transfer process bringing the for the CL reaction (usually an oxidant); some species reactants together. The principal advantages of this system that affects the rate or efficiencies of CL reaction, lie in the conservation of reagents and the convenience of activators such as catalyst (enzymes or metal ions) measurement. or inhibitors such as reductants that inhibit the CL The third approach involves the use of flow measurement emission; fluorphores in the case of sensitized CL, systems. The flow injection approach has been described as some species that are not directly involved in the the most successful of the methods. It involves injection of CL reaction but that can react with other reagents the analyte into a steam of appropriate pH, remote from in coupled reactions to generates a product that is the detector, and the chemiluminescent reagent flows in a reactant in the CL reaction, species that can be another stream. The two streams meet at a T-junction inside derivatized with some CL precursors or fluorophores a light-tight enclosure, then flow through a flat coil placed being determined by direct or sensitized CL immediately in front of a photomultiplier tube. This compact (5) Depending on the nature of the analyte and the CL assembly provides more rapid and reproducible mixing, reaction the increase or decrease of CL intensity will resulting in reproducible emission intensities and permitting be directly related to the analyte concentration. rapid sample through put. The design of the mixing device and the means of retaining the emitting solution in view of the (6)CLreactionscanbecoupledasadetectiontech- detectors are of important consideration. Mixing is reported nique in chromatography, capillary electrophoresis, as being the most effective at a T-piece or a Y-junction, or immunoassay, providing qualitative and/or quan- although some have used a conventional FIA in which sample titative information or a large variety of species in the is injected in to the surrounding flowing reagent to achieve gas and liquid phases. mixing. This approach is reproducible but reported as not (7) CL reaction can be coupled as a detection tech- producing a rapid mixing. In flow measurement system, the nique in chromatography, capillary electrophoresis or chemiluminescence signal has to be measured during the immunoassay, providing quantitative and/or quanti- mixing:asaresult,onlyasectionasopposedtothewhole tative information of a large variety of species in the intensity-time curve is measured. An additional feature of gas and liquid phase. these systems is that the shape of the curve now depends on both the kinetics of the chemilluminercent reaction and As an empirical rule, CL behavior may be predicated in the parameters of the flow system. Chemical variables such a compound or its derivatization product has fluorescence as reagent concentration and pH, and physical parameters properties. It is possible that oxidization of such a species may such as flow rate, reaction coil length, sample size, and the produce CL, but there are many exceptions to this general rule limitations of experimental apparatus all affects the perfor- [49–51]. mance of flow injection procedures. The effects of these onthe observed analytical signal are not necessarily independent, as 3.1. Instrumentation. The instrumentation employed for interactions can and probably do occur [52, 53]. chemiluminescence measurements basically consists of a The sample and the reagent could be either in continuous mixing device and a detection system. Three approaches have flow or in stopped flow approach. Continues flow CL systems been used to measure the intensity of emitted light. are used in gas phase and solution phase reactions. The The first approach involves the use of static measurement sample and CL reagents are continuously pumped and mixed system in which the mixing of reagents is performed in a together using a merging zones of flow injection manifold and vessel held in front of the detector. The chemiluminescence senttoaflowcellormixedandtheemissioninanintegral reagentisaddedtotheanalyteinacuvetteheldina reactor flow cell. The signal is observed when the cell is totally dark enclusure, and the intensity of the light emitted is filled with the reaction mixture, at a fixed period after mixing. measured through an adjacent photomultiplier tube. In this In this case, it is possible to obtain a constant and reproducible static system, the mixing is induced by the force of the signal, easier to measure, which represents the integrated injection. This simple reagent addition system without a output over the residence time of the reaction mixture in mixing device is of limited usefulness in measuring fast the cell. Optimum sensitivity is achieved by adjusting the chemiluminescent reactions with precision. The procedure is flow rate, the observation cell volume, and transfer channel also rather cumbersome, requiring separate cuvettes for each volumes, with the aim of observing portion of emission measurements as well as repeated opening of the light-tight profile to occur at the maximum of the CL intensity- versus apparatus, which necessitates special precautions to protect time profile. Disadvantage of this approach include the high the photomultiplier tube. reagent consumption and the fact that no kinetic information The second approach is the two-phase measurement is obtained and that only a very small portion of the total CL system. In this system, the chemilluminercent reagents are intensity is measured for slow reactions (Figure 5). immobilized on a solid support such as filter paper, and the Stopped flow approach has interesting features for CL analyte is permitted to interact with the immobilized reagent monitoring in very fast CL reactions. In this case, the sample by diffusion or convection. The light emitted is measured and reagent are efficiently mixed in a reaction chamber and ISRN Spectroscopy 9

ICL

Relative units

Max (t1)

Or area in At I Reaction cell max

Detector

t t 0 1 Reaction time Concentration (s) (a) (b) (c)

Figure 5: Basic steps in a CL process: (a) the sample and reagent(s) are introduced is the reaction cell and the final reagent is injected to initiate the CL emission, then lights is monitored by the detector, (b) curve showing CL intensity as a function of time after reagents mixing to initiate the reaction (the decay of the signal is due to the consumption of reagents and changes in the CL quantum efficiency with time); (c) a calibration function is established in relation to increasing analyte concentrations. rapidly forced to an observation cell, in which the flow is or by absorption, and it can interact with the analyte by violently stopped, allowing monitoring of the full intensity- diffusion or convection. In gas phase CL reaction, the sample time curve and allowing kinetics measurements (Figure 5) and the CL reagents flow inside a reactor through calibrated which can be related to the analyte concentration with a leaks and the CL emission produced and monitored by higher precision and selectively than using peak height or the detector through and optical window. Commercial and areas. homemade luminormeters for liquid, solid, and gas phase analysis use various cell configurations. 3.1.1. Reaction Cell. Depending on the CL reaction that is carried out in liquid, solid or gas phases, it is possible to use 3.1.2. Detector. There are four basic requirements for the different reaction cells, the essential requirements for their detectors. Which constitute the critical parts of the lumi- design being that the maximum intensity must be reached normeter. while the stream containing the analyte and the CL reagents (1)Itmustbeabletodetectalightsignaloverseveral is in front of the detector. It is a very critical variable because orders of magnitude of intensity form just a few in both static and flow methods, the magnitude of the CL photons per second to tens of millions photons per intensity is proportional to the volume of the cell. Usually it second. is possible to apply any material that transmits light in the visible range and that is compatible with the sample, such as (2)Itmustbesensitiveatleastoverthespectralrange glass, quartz, or even acrylic or other plastics. Polystyrene is 400–600 nm and ideally over the complete range of not recommended because it can build up static electricity, the visible spectrum (i.e., 380–750 nm) and even over which may contribute to elevated background levels. More the UV and IR regions. When its sensitivity varies transparent cell materials allow more light to reach the with wavelength, as is usually the case, it must be detector, resulting in better detection limits. possible to correct the data in this respect. In the case of flow CL measurements in the liquid phase, (3) The signal output presented by the detector should the cell design most frequently used is a long spiral cell be directly related to the light intensity reaching it, (about 1 cm) which is located as close to the detector as ideally linearly over the entire sensitively range. The possible. The large volume is required due to the need to signal from the detector should be produced in a form collect a greater amount of emitted light and because the thin thatcanbeeasilydisplayedrecordedandanalyzed. 𝜇 size required (50–100 m) minimizes band broadening and (4) The speed of response of the detector must be much provides a thin optical path to reduce inner filtering effects faster than the rate of the CL reaction, if the signal of the CL emission. With the purpose of minimize sample output is not to be a distorted version of the true dispersion, usually, the injection valve and the cell must be signal. placedasclosetoreachtheotheronesaspossible. In heterogeneous systems, the reagents can be immobi- Light is detected by photosensitive devices through the lized on a solid surface either covalently, behind a membrane, generation of photocurrent. Photomultiplier tubes (PMT) are 10 ISRN Spectroscopy the detection devices more frequently used in CL because on steroid immunoassay. In this case a synthetic acridinium of their high gains making them suitable for low light level derivative of estradiol was prepared which could be detected detection. However they must be operated with very stable with high sensitivity. Assay performance was however limited power supplies to keep the total gain constant. There are two by the low affinity of the antibody for the labelled derivative different configurations for PMTs depending on their posi- once again illustrating the dependence of hapten systems on tions in relation to the reaction cell: either to the side (“side- suitable immunochemistry. on” configuration) or underneath the reaction cell (“end-on” Theprocedurehascentredonthedevelopmentofassay configuration). Side on PMTs is usually more economical methods for polypeptides using acridinium ester labelled than end on models and their vertical configuration takes up antibodies. It has employed almost exclusively two-site assay less space than be end on version. However, end-on PMTs procedures in which the analyte is reacted either sequentially have large photocathode area and offer greater uniformity of or simultaneously with labeled antibodies and solid-phase light collection and of the response. antibodies. In this procedure, the uptake of labelled antibody Also in the absence of light stimulation all practical on to solid-phase antibody is a function of the quantity of ana- light detectors produce an unwanted output signal termed lyteboundandhencetheanalyteconcentrationinthesample. “background” which is referred to as “dark counts” or dark The most satisfactory solid-phase antibodies developed so current” depending on the applications. At moderate bias, far are prepared by reacting antibody preparations with the voltages, dark current can be due primarily to thermal diazonium salt of re-precipitated aminoaryl cellulose. This emission of electrons at the photocathode and from the preparation has the advantage that it remains in suspension dynodes. This undesirable signal can be reduced by cooling during the reaction without agitation. It is also extremely ∘ the PMT using temperature of 60 to 0 C. efficient as an immune adsorbent since it has a high binding capacity for protein as well as very low non-specific uptake of protein [54, 55]. 3.2. Pharmaceutical Application of Chemiluminescence Reac- In this procedure standards or serum samples (100 pi) are tion. From Table 1 one can observe that varsitile compounds reacted with a monoclonal antibody (100 p1, 1.5 ng) labelled and pharmaceuticals could be directly or indirectly quanti- with 3 mol/mol acridinium ester. After 2 h, solid-phase anti- fied. For the indirect method of quantification either chemi- body suspension (100 p1, 50 Pg) is added and after a further cally derived so as to exhibit the compound luminescent effect 1 h, 1 mL of buffer is added and the mixture centrifuged. or by measuring the compounds quenching effect. After one further 1 mL wash, the chemiluminescent activity associated with the solid-phase is quantified luminometri- 3.3. Chemiluminescent Immunoassay. One of the most sig- cally following injection of 200pl water to resuspend the nificantapplicationsofchemiluminescentmoleculeshas pellet followed by 200 p1 0.1% (v/v) H2O2 in 0.1 M NaOH. been labels in immunoassay. Not unexpectedly the con- Thephotoncountsareintegratedovera2speriodandthe straints arise from the need to couple the chemiluminescent bound counts related to the dose of analyte present. The use molecules to the analytes of respective antibodies in such of reagent excess methodology together with an acridinium a way that the properties of the label are not disturbed. In ester label of high specific activity combine to make this the particular aminobutyl ethyl isoluminol (ABEl) which can be most sensitive immunoassay yet described for TSH [56, 57]. coupled to low molecular weight compounds without loss in From the clinical point of view this assay offers a remark- quantum yield. able breakthrough in diagnostic and therapeutic testing. Asaresult,ABElandrelatedmoleculeshavebeenusedas Pituitary TSH secretion plays a central role in the control of labels for a variety of steroid immunoassays which have levels thyroid function. Since it is under negative feedback control of performance comparable to those of the corresponding by the thyroid hormones whose production it stimulates, it radioimmunoassay. A universal problem of these methods provides a sensitive indicator of thyroid status. arises from the extensive quenching of the signal when The main applications of TSH measurement being in the the label is associated with antibody. So great is the loss confirmation of hypothyroidism where levels are elevated and in quantum yield under these conditions that dissociation in monitoring the pituitary response to thyrotropin release of the complex prior to luminometry has been adopted hormone (TRH). This immune-chemiluminometric assay as an obligatory step. As indicated above, the majority of (ICMA) is at present under evaluation [58]. chemiluminescent hapten immunoassays so far described have been based on ABEl or similar labels. These assays include plasma estradiol-l7, testosterone, and progesterone References as well as urinary conjugated steroids such as pregnanediol- [1] L. Bøtter-Jensen, “Luminescence techniques: instrumentation 3n-glucuronide and estrone glucuronide. Dissociation of the and methods,” Radiation Measurements,vol.27,no.5-6,pp. label prior to signal detection has been facilitated by means 749–768, 1997. of strong alkali often at high temperature. Though this results in satisfactory light emission, it does introduce a step into [2] Y. Hajime, Nitride Phosphors and Solid-State Lighting,Taylor& the analytical procedure which is operationally complex and Francis, 2011. which may affect the overall performance of the assay itself. [3] K. H. 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